Chromogens are molecules that allow detection of a target using enzyme-based precipitation reactions and their use in immunohistochemistry (IHC) allows visualization of the immune complex (and hence the antigen) in the context of tissue architecture. The most widely used chromogenic compound, 3,3′-diaminobenzidine (DAB), provides brown-colored staining, also commonly called “brown staining” (diaminobenzidine chromogenic staining or immunostaining). Optimal chromogenic staining relies on the deposition of a sufficient amount of substrate to block light. In the case of DAB, a “desirable’ image” is regarded to be produced when deposition of the substrate leads to an absorbance of 1-2 units, meaning that 90 to 99% of the light signal is blocked. Although this creates a contrast that is easy to read, it hampers the use of multiple colocalized chromogens on routine assays (Carvajal-Hausdorf et al., 2015, Laboratory investigation, 95, 385-396).
Chromogenic IHC is omnipresent in cancer diagnosis but has been criticized for its technical limit in quantifying the level of protein expression on tissue sections, thus potentially masking clinically relevant data. Historically, the use of brown staining aimed at determining the presence or absence of a biomarker rather than intensity level of the staining. In fact, high levels of signal (staining) were even aimed at in order to ease the readings.
However, with the development of assays and treatments, assay read-outs no longer aim at a simple binary answer (expressed or not) but the quantification of the expression amount has also become a critical variable which is of high importance in the field of biomarkers, evolving from diagnostics to prediction of response to therapy. In this context, the limitations of immunodiagnostic assays have become more important (Rimm et al., 2006, Nature Biotechnology, 24, 914-916).
In particular, with the advancement of personalized cancer medicine, precise molecular profiling of tumors is gaining significant importance in routine diagnostic pathology and with the evolution towards personalized treatments tailored to the molecular features of malignant tumors, the last decade has witnessed an increasing use of molecular analysis approaches, including but not limited to in situ hybridization (ISH), mRNA expression profiling techniques and next generation sequencing (NGS). Immunohistochemistry (IHC), however, remains by far the most used method in the routine diagnostic evaluation of tumor tissues, with the advantages of wide availability, low cost, and preservation of the information-rich morphological context.
Continuous quantification of protein expression in tumor sections has long been the missing link between methods analyzing nucleic acids and conventional IHC. The majority of IHC tests currently used in clinical diagnosis cannot quantify the antigen (Ag) expression but rather perform a binary or semi-quantitative assessment as interpreted by the pathologist. An example of such semi-quantitative tests is the assessment of epidermal growth factor receptor 2 (HER2) protein expression level in breast cancer (known to promote the growth of cancer cells), for which the scoring can have four different levels: 0, 1+, 2+ or 3+(Wolf et al., 2013, J. Clin. Oncol., 31, 3997-4013). Therefore, this non-continuous assessment results in a loss of information regarding the Ag expression level (Rimm, 2006, supra; Carvajal-Hausdorf et al., 2015, supra). Furthermore, it suffers from possible ambiguous, equivocal results and relative subjectivity in scoring between different pathologists.
To overcome the discrete nature of the semi-quantitative scoring methods, more continuous scoring algorithms such as the “H-score” have been proposed (Detre et al., 1995, J. Clin. Pathol., 48, 876-878). These methods involve manually assessing the approximate sample areas with different levels of target biomarker expression (e.g. HER2) and multiplying the areas with appropriate “weights” according to the expression amount. However, this approach has other drawbacks such as the inability of the scorer to detect subtle differences in target expression especially at the low and high ends of the scale and the tendency to round scores, effectively converting the approach to another semi-quantitative one (Camp et al., 2002, Nature Medicine, 8, 1323-1328).
Techniques such as Western Blot and ELISA provide means for protein quantification but at the cost of the loss of morphological information and the integrity of the samples of interest since they require the lysing of the samples (Becker et al., 2007, J. Pathol., 211, 370-378). While they potentially offer high reproducibility and accuracy, they are either regarded as complementary methods or utilized in assays for liquid media such as detection of circulating tumor elements in serum and therefore are less suitable for assessing protein expression levels in the morphological context of the tissue slide.
Genetic methods, particularly Fluorescent In-Situ Hybridization (FISH), are widely utilized as complementary techniques in cases of inconclusive results obtained with IHC or similar tests. Metrics such as the gene copy number provide quantitative information on the biomarker of interest. On the other hand, FISH is relatively expensive. Furthermore, the existence or amplification of a gene is a necessary but not a sufficient condition for the expression of a diagnostically relevant antigen biomarker. Comparison studies between IHC and FISH methods for HER2 have been widely performed in clinical research (Pauletti et al., 2000, J. Clin. Oncol., 18, 3651-3664; Owens et al., 20014, Clin. Breast Cancer, 5, 63-69) and studies conclude that agreement between IHC and FISH is not complete, especially for IHC 2+ cases, creating issues with false positives and false negatives.
In this context, as clinical pathology moves from qualitative to quantitative, immunofluorescence (IF) is gaining relevance in the research settings and laboratory-developed tests, mainly due to its increased capacity to measure the signal intensity of one or more biomarkers as compared to traditional chromogenic techniques (Carvajal-Hausdorf et al., 2015, supra). Several image processing techniques that quantify the extent of IF signal have already been reported in the literature (McCabe et al., 2005, JNCI J. Natl. Cancer Inst., 97, 1808-1815; Rojo et al., 2010, Folia Histochem. Cytobiol. 47). However, there is little or no evidence suggesting that the IF signal per se can be used to precisely quantify Ag expression amount on tissue sections. Indeed, due to the kinetics of Ag-antibody (Ab) binding, a 2-step IF assay does not result in a signal that is linearly proportional to the Ag expression (Caelen et al., 2000, Langmuir, 16, 9125-9130; Squires et al., 2008, Nat. Biotechnol., 26, 417-426), which potentially ends up in a misleading quantification and, hence, obscures the potential of IF in providing precise biomarker data.
Therefore, shifting from qualitative to quantitative, immuno fluorescence (IF) has recently gained attention, yet the question of how precisely IF can quantify antigen expression remains unanswered, regarding in particular its technical limitations and applicability to multiple markers. There is therefore a need to find precise methods that allow to routinely and precisely quantify biomarker expression in tissues while preserving the morphology since those methods would not only reduce the requirement for expensive complementary gene analysis but also increase the precision of diagnosis, prognosis and the success of targeted therapies, in clinical trials and routine patient care.